N-acetylglucosamine transferase, nucleic acid encoding the same and use thereof in diagnosing cancer and/or tumor

ABSTRACT

An enzyme having an activity of transferring N-acetylglucosamine to the non-reducing end of a Galβ1-4Glc or Galβ1-4GlcNAc-group via a β-1,3 bond; a nucleic acid encoding the same; and a method of diagnosing cancer and/or tumor, in particular, digestive cancer and/or tumor using the expression dose of a gene of the above enzyme as an indication. A gene of a novel enzyme having an activity of transferring N-acetylglucosamine to the non-reducing end of a Gal β1-4Glc or Gal β1-4GlcNAc-group via a β-1,3 bond is cloned from human stomach cells and its base sequence is determined. Then this enzyme is expressed. Since this enzyme is scarcely or never produced in cancer and/or tumor, in particular, digestive cancer and/or tumor cells, cancer and/or tumor can be diagnosed with the use of the expression of the enzyme gene as an indication.

TECHNICAL FIELD

The present invention relates to a novel enzyme having an activity totransfer N-acetylglucosamine to a non-reducing terminal of Galβ1-4Glc orGalβ1-4GlcNAc group through β1,3-linkage, and to a nucleic acid codingfor the same, as well as to nucleic acids for measuring the nucleicacid. The present invention further relates to diagnosis of cancer ortumor using the expression amount of the above-mentioned enzyme or thegene thereof as an index.

BACKGROUND ART

Five types of enzymes are known, having an activity to transferN-acetylglucosamine to a non-reducing terminal of Galβ1-4Glc orGalβ1-4GlcNAc group through β1,3-linkage, which activity is involved inthe synthesis of polylactosamine sugar chains (Togayachi, A. et al., JBiol Chem, 2001, 276, 22032–40; Shiraishi, N. et al., J Biol Chem, 2001,276, 3498–507; Sasaki, K et al., Proc Natl Acad Sci USA, 1997, 94,14294–9). However, although the amount of polylactosamine on cellsurfaces is increased by making the cells express the gene of theenzyme, some of the enzymes expressed have very low activities. Thus,although it is thought that the enzymes which produce polylactosaminehave different characteristics, the characterization of the enzymes hasnot been sufficient. Therefore, to prepare or produce thepolylactosamine sugar chain structure which requires the enzymeactivity, it is necessary to chemically synthesize the structure,isolating the structure from a biological component or to synthesize thestructure enzymatically using a tissue homogenate.

It is known that sugar chain structures such as Lewis antigen exist onthe sugar chain structures based on polylactosamine sugar chains(Kannagi R. Glycoconj J. 1997 August; 14(5):577–84. Review; Nishihara Set al., J Biol. Chem. 1994 Nov. 18; 269(46):29271–8). Similarly, it issaid that the structures such as the lengths of polylactosamine sugarchains are involved in cellular immunity by NK cells or the like (OhyamaC et a., EMBO J. 1999 Mar. 15; 18(6):1516–25). Similarly, it is knownthat human stomach tissue is infected with Helicobacter pylori through arelated sugar chain such as Lewis antigen (Wang G et al., Mol Microbiol.2000 June; 36(6):1187–96. Review; Falk PG et al., Proc Natl Acad SciUSA. 1995 Feb. 28; 92(5):1515–9). Thus, if the gene of an enzyme havingan activity to transfer N-acetylglucosamine to a non-reducing terminalof Galβ1-4Glc or Galβ1-4GlcNAc group through β1,3-linkage can be cloned,and if the enzyme can be produced by a genetic engineering process usingthe gene, an antibody to the enzyme may also be produced. Therefore,these are useful for the diagnoses, therapies and prophylactics ofcancers, immune diseases and infectious diseases by pylori. However, theenzyme has not yet been purified or isolated, and there is no clue tothe isolation of the enzyme and identification of the gene. As a result,an antibody to the enzyme has not been prepared.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide an enzymehaving an activity to transfer N-acetylglucosamine to a non-reducingterminal of Galβ1-4Glc or Galβ1-4GlcNAc group through β1,3-linkage, anda nucleic acid coding for the same. Another object of the presentinvention is to provide a recombinant vector which expresses theabove-mentioned the nucleic acid in a host cell, to provide a cell inwhich the nucleic acid is introduced and which expresses the nucleicacid and the enzyme protein, and to provide the enzyme protein. Stillanother object of the present invention is to provide a nucleic acid formeasurement of the above-mentioned nucleic acid according to the presentinvention, and to provide a method for producing the enzyme having theactivity.

As mentioned above, since the enzyme of interest has not been isolated,it is impossible to know its partial amino acid sequence. In general, itis not easy to isolate and purify a protein contained in cells in atrace amount, and so isolation of the enzyme from cells, which has notbeen isolated so far, is expected not easy. The present inventorsthought that if there is a homologous region among the nucleotidesequences of the various enzyme genes, which enzymes have relativelysimilar actions to that of the enzyme of interest, the gene of theenzyme of interest may also have the homologous sequence. Aftersearching the nucleotide sequences of the knownβ1,3-N-acetylglucosaminyltransferase genes, β1,3-galactoslytransferasegenes and β1,3-N-acetylgalactosaminyltransferase genes, a homologousregion was discovered. Thus, based on the cloning by PCR using cDNAlibrary, in which a primer was set in the homologous region, and aftervarious considerations, the present inventors succeeded in the cloningof the gene of the enzyme, and its nucleotide sequence and the deducedamino acid sequence were determined, thereby accomplishing the presentinvention.

That is, the present invention provides a protein having the amino acidsequence shown in SEQ ID NO: 1 in SEQUENCE LISTING, or a protein havingthe same amino acid sequence as shown in SEQ ID NO:1 except that one ormore amino acids are substituted or deleted, or that one or more aminoacids are inserted or added, which has an activity to transferN-acetylglucosamine to a non-reducing terminal of Galβ1-4Glc orGalβ1-4GlcNAc group through β1,3-linkage. The present invention alsoprovides a nucleic acid coding for the protein. The present inventionfurther provides a recombinant vector containing the nucleic acid, whichcan express the nucleic acid in a host cell. The present invention stillfurther provides a cell which is transformed by the recombinant vector,which expresses the nucleic acid. The present invention still furtherprovides a nucleic acid for measurement of the nucleic acid, whichspecifically hybridizes with the nucleic acid. The present inventionstill further provides use of the nucleic acid for measurement for thediagnosis of a cancer or tumor. The present invention still furtherprovides a method for diagnosis of a cancer or tumor, comprisingdetermining the amount of the above-mentioned enzyme or determining theexpression amount of the gene coding for the enzyme, in (a) samplecell(s) separated from body. The present invention still furtherprovides a method for measuring the above-mentioned nucleic acidaccording to the present invention, comprising annealing the nucleicacid for measurement of nucleic acid, according to the presentinvention, and the above-described nucleic acid according to the presentinvention so as to hybridize them, and measuring the hybridized nucleicacid. The present invention still further provides use of the nucleicacid for measurement of nucleic acid, according to the presentinvention, for the production of nucleic acid for measurement of nucleicacid according to the present invention. The present invention stillfurther provides use of the nucleic acid for measurement of nucleicacid, according to the present invention, for the production ofdiagnostic reagent for a cancer and/or tumor.

By the present invention, an enzyme having an activity to transferN-acetylglucosamine to a non-reducing terminal of Galβ1-4Glc orGalβ1-4GlcNAc group through β1,3-linkage, and a nucleic acid encodingthe enzyme were first provided. Further, by the present invention, anucleic acid for measuring the above-mentioned nucleic acid was firstprovided. Still further, a simple and accurate method for diagnosis of acancer or tumor, especially a cancer or tumor of digestive organs, and anucleic acid for measurement used therefor were first provided. Thus, itis expected that the present invention will greatly contribute to thediagnoses of cancers and tumors of digestive organs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of the flow cytometry showing the bindingproperty between the HCT15 colon cancer cell line and the LEA lectin,the cell line being transformed with a recombinant vector into which thegene of the present invention was incorporated or with a recombinantvector into which the gene of the present invention was notincorporated.

FIG. 2 shows the results of the flow cytometry showing the bindingproperty between the LSC colon cancer cell line and the LEA lectin, thecell line being transformed with a recombinant vector into which thegene of the present invention was incorporated or with a recombinantvector into which the gene of the present invention was notincorporated.

FIG. 3 shows the results of the flow cytometry showing the bindingproperty between the HCT15 colon cancer cell line and the WGA lectin,the cell line being transformed with a recombinant vector into which thegene of the present invention was incorporated or with a recombinantvector into which the gene of the present invention was notincorporated.

FIG. 4 shows comparison of the amount of expression of the geneaccording to the present invention in normal tissues and that in cancertissues of colon cancer patients.

BEST MODE FOR CARRYING OUT THE INVENTION

The nucleic acid resulting from the removal of the initiation codon(ATG) from the nucleic acid encoding the protein of the presentinvention, which was cloned from a human antrum cDNA library by themethod that will be described in detail in the Examples below, has thenucleotide sequence shown in SEQ ID NO: 4 in the SEQUENCE LISTING, andthe deduced amino acid sequence encoded thereby is described below thenucleotide sequence. In SEQ ID NO:3, the amino acid sequence alone isshown. In the Examples below, the nucleic acid having the nucleotidesequence shown in SEQ ID NO:4 was incorporated into an expressionvector, expressed in insect cells and it was confirmed that a proteinhaving the above-mentioned enzyme activity was produced. By comparingthe amino acid sequence shown in SEQ ID NO:3 and the amino acid sequenceof a similar enzyme (concrete enzyme name: β3GnT2: AB049584 which is thegene of β-1,3-N-acetylglucosaminyltransferase), it is thought that theregion with a relatively high homology, that is, the region from the45th amino acid to the C-terminal of the amino acid sequence shown inSEQ ID NO:3 is the active domain of the enzyme, and that theabove-mentioned enzyme activity is exhibited if this region consistingof 283 amino acids is contained. This 283 amino acids is shown in SEQ IDNO:1 and the nucleic acid encoding this, taken out from SEQ ID NO:4, isshown in SEQ ID NO:2.

The protein (named “β3GnT-7”) according to the present inventionobtained in the Examples below is an enzyme having the followingcharacteristics. Each of the characteristics as well as the methods formeasuring them are described in detail in the Examples below.

-   Action: Transfer N-acetylglucosamine to a non-reducing terminal of    Galβ1-4Glc group or Galβ1-4GlcNAc group through β1,3-linkage. The    reaction catalyzed by the enzyme, expressed in terms of reaction    equation, is as follows:    UDP-N-acetyl-D-glucosamine+β-D-galactosyl-1,4-D-glucosyl-R→UDP+N-acetyl-β-D-glucosaminyl-1,3-β-D-galactosyl-1,4-D-glucosyl-R,    or    UDP-N-acetyl-D-glucosamine+β-D-galactosyl-1,4-N-acetyl-D-glucosaminyl-R→UDP+N-acetyl-β-D-glucosaminyl-1,3-β-D-galactosyl-1,4-N-acetyl-D-glucosaminyl-R-   Substrate Specificity: Galβ1-4Glc group or Galβ1-4GlcNAc group. In    biological substances, these groups occurs abundantly as, for    example, polylactosamine structures in glycoproteins (O-glycans and    N-glycans) and glycolipids (lacto•neolacto series sugar chains and    the like). Further, the Galβ1-4Glc groups or Galβ1-4GlcNAc groups    contained in the basal structures of proteoglycans (keratan sulfate)    and the like.

In general, it is well-known in the art that there are cases wherein thephysiological activity of a physiologically active protein such as anenzyme is retained even if the amino acid sequence of the protein ismodified such that one or more amino acids in the amino acid sequence issubstituted or deleted, or one or more amino acids are inserted or addedto the amino acid sequence. Therefore, a protein having the same aminoacid sequence as shown in SEQ ID NO:1 or 3 except that one or more aminoacids are substituted or deleted, or one or more amino acids areinserted or added, which protein has an activity to transferN-acetylglucosamine to a non-reducing group of Galβ1-4Glc orGalβ1-4GlcNAc group through β1,3-linkage (the protein is hereinafterreferred to as “modified protein” for convenience) is also within thescope of the present invention. The amino acid sequence of such amodified protein preferably has a homology of not less than 70%,preferably not less than 90%, still more preferably not less than 95% tothe amino acid sequence shown in SEQ ID NO: 1 or 3. The homology of thenucleotide sequence may easily be calculated by using a well-knownsoftware such as FASTA, and such a software is available on theinternet. Further, as the modified protein, one having the same aminoacid sequence as shown in SEQ ID NO:1 or 3 except that one or severalamino acids are substituted or deleted, or that one or several aminoacids are inserted or added is especially preferred. Further, a proteincontaining the protein having the amino acid sequence shown in SEQ IDNO:1 or 3, or a modified protein thereof, which has an activity totransfer N-acetylglucosamine to a non-reducing terminal of Galβ1-4Glc orGalβ1-4GlcNAc group through β1,3-linkage is also within the scope of thepresent invention. For example, in the Examples below, a nucleic acidencoding a membrane-bound type enzyme, in which a transmembrane regionis ligated to the upstream of the amino acid sequence shown in SEQ IDNO:3 was also cloned, and such a membrane-bound type enzyme is alsowithin the scope of the present invention.

The present invention also provides nucleic acids coding for the aminoacid sequence shown in SEQ ID NO:1 or 3 and nucleic acids coding for theamino acid sequences of the above-mentioned modified proteins. As thenucleic acid, DNA is preferred. As is well-known, due to degeneracy,there may be a plurality of codons each of which codes for the samesingle amino acid. However, as long as a nucleic acid codes for theabove-described amino acid sequence, any nucleic acid having anynucleotide sequence is within the scope of the present invention. Thenucleotide sequences of the cDNA actually cloned in the Examples beloware shown in SEQ ID NOs:2 and 4. Those nucleic acids which hybridizewith the nucleic acid having the nucleotide sequence shown in SEQ IDNO:2 or 4 under stringent conditions (i.e., hybridization is performedat 50 to 65° C. using a common hybridization solution such as 5×Denhardt's reagent, 6×SSC, 0.5% SDS or 0.1% SDS), and which code for theabove-described modified proteins are within the scope of the presentinvention.

The above-described nucleic acid according to the present invention canbe prepared by the method described in detail in Example below.Alternatively, since the nucleotide sequence was clarified by thepresent invention, it can easily be prepared by using human antrum asthe material and performing the well-known RT-PCR method. Theabove-described protein according to the present invention can also beeasily prepared by, for example, incorporating the above-describednucleic acid according to the present invention into an expressionvector, expressing the nucleic acid in a host cell, and purifying theproduced protein.

By inserting the above-described nucleic acid according to the presentinvention into a cloning site of an expression vector, a recombinantvector which can express the above-described nucleic acid in a host cellmay be obtained. As the expression vector, various plasmid vectors andvirus vectors for various host cells are well-known and commerciallyavailable. In the present invention, such a commercially availableexpression vector may preferably be employed. The methods fortransforming or transducing host cells with such a recombinant vectorare also well-known. The present invention also provides a cell intowhich the nucleic acid according to the present invention is introducedby transformation, transduction or transfection, which expresses thenucleic acid. The methods per se for introducing a foreign gene into ahost cell are well-known, and the introduction of the foreign gene mayeasily be attained by, for example, using the above-mentionedrecombinant vector. An example of the construction of a recombinantvector and a method for introducing the nucleic acid according to thepresent invention into host cells using the recombinant vector aredescribed in detail in the Examples below.

Sugar chains may be bound to the protein according to the presentinvention, as long as the protein has the amino acid sequence describedabove and has the above-described enzyme activity. In other words, theterm “protein” used herein also includes “glycoprotein”.

Since the nucleotide sequence of the cDNA of the novel enzyme accordingto the present invention was clarified by the present invention, nucleicacids for measurement according to the present invention (hereinafterreferred to as simply “nucleic acid for measurement”), whichspecifically hybridize with the mRNA or the cDNA of the enzyme, wereprovided by the present invention. The term “specifically” herein meansthat the nucleic acid does not hybridize with other nucleic acidsexisting in the cells subjected to the test and hybridizes only with theabove-described nucleic acid according to the present invention.Although it is preferred, in general, that the nucleic acid formeasurement has a sequence homologous with a part of the nucleic acidhaving the nucleotide sequence shown in SEQ ID NO:2 or 4, mismatch ofabout 1 or 2 bases does not matter in many cases. The nucleic acid formeasurement may be used as a probe or a primer in a nucleicacid-amplification method. To assure specificity, the number of bases inthe nucleic acid for measurement is preferably not less than 15, morepreferably not less than 18. In cases where the nucleic acid is used asa probe, the size is preferably not less than 15 bases, more preferablynot less than 20 bases, and not more than the full length of the codingregion. In cases where the nucleic acid is used as a primer, the size ispreferably not less than 15 bases, more preferably not less than 18bases, and less than 50 bases. The methods for measuring a test nucleicacid using a nucleic acid having a sequence complementary to a part ofthe test nucleic acid as a primer of a gene-amplification method such asPCR or as a probe are well-known, and the methods by which the mRNA ofthe enzyme according to the present invention was measured by Northernblot or in situ hybridization are concretely described in detail in theExamples below. In the present specification, “measurement” includesdetection, quantification and semi-quantification.

The nucleic acid-amplification methods such as PCR are well-known in theart, and reagent kits and apparatuses therefor are commerciallyavailable, so that they may easily be carried out. That is, for example,a test nucleic acid serving as a template (e.g., the cDNA of the gene ofthe enzyme of the present invention) and a pair of nucleic acids formeasurement (primers) according to the present invention are mixed in abuffer in the presence of Taq polymerase and dNTPs, and the steps ofdenaturation, annealing and extension are carried out by changing thetemperature of the reaction mixture. Usually, the denaturation step iscarried out at 90 to 95° C., the annealing step is carried out at Tmbetween the template and the primers or a vicinity thereof (preferablywithin ±4° C.), and the extension step is carried out at 72° C. which isthe optimum temperature of Taq polymerase. The reaction time of eachstep is selected from about 30 seconds to 2 minutes. By repeating thisthermal cycle for about 25 to 40 times, the region between the pair ofprimers is amplified. The nucleic acid-amplification method is notrestricted to PCR, but other nucleic acid-amplification methodswell-known in the art may also be employed. By carrying out the nucleicacid-amplification method using a pair of the above-described nucleicacids for measurement according to the present invention as primers andusing the test nucleic acid as a template, the test nucleic acid isamplified. In contrast, in cases where the test nucleic acid is notcontained in the sample, the amplification does not occur. Therefore, bydetecting the amplification product, whether the test nucleic acidexists in the sample or not may be determined. Detection of theamplification product may be carried out by a method in which thereaction solution after the amplification is subjected toelectrophoresis, and the bands are stained with ethidium bromide or thelike, or by a method in which the amplification product afterelectrophoresis is immobilized on a solid phase such as a nylonmembrane, a labeled probe which specifically hybridizes with the testnucleic acid is hybridized with the test nucleic acid, and the labelafter washing is detected. Alternatively, the test nucleic acid in thesample may be quantified by the so called realtime detection PCR using aquencher fluorescent pigment and a reporter fluorescent pigment. Sincethe kits for realtime detection PCR are also commercially available,realtime detection PCR may also be carried out easily. The test nucleicacid may also be semi-quantified based on the intensity of the bandresulted in electrophoresis. The test nucleic acid may be a mRNA or acDNA reverse-transcribed from a mRNA. In cases where a mRNA is amplifiedas the test nucleic acid, NASBA method (3SR method, TMA method) usingthe above-described pair of primers may also be employed. NASBA methodper se is well-known, and kits therefor are commercially available, sothat NASBA method may easily be carried out using the above-describedpair of primers.

As the probe, labeled probe obtained by labeling the above-describednucleic acid for measurement with a fluorescent label, radioactivelabel, biotin label or the like may be used. The methods per se forlabeling a nucleic acid are well-known. Whether the test nucleic acidexists in the sample or not may be determined by immobilizing the testnucleic acid or amplification product thereof, hybridizing the labeledprobe therewith, and measuring the label bound to the solid phase afterwashing. Alternatively, the nucleic acid for measurement is immobilized,the test nucleic acid is hybridized therewith, and the test nucleic acidbound to the solid phase is detected by a labeled probe or the like. Insuch a case, the nucleic acid for measurement immobilized on the solidphase is also called a probe. The methods for measuring a test nucleicacid using a nucleic acid probe are also well-known in the art, and maybe attained by making contact between the nucleic acid probe and thetest sample in a buffer at Tm or a vicinity thereof (preferably within±4° C.) so as to hybridize them, and then measuring the hybridizedlabeled probe or the test nucleic acid bound to the immobilized probe.Such a method includes well-known methods such as Northern blot and insitu hybridization described in the Examples below, as well as Southernblot.

By making the enzyme according to the present invention act on aglycoprotein, oligosaccharide or polysaccharide having (a) Galβ1-4Glc orGalβ1-4GlcNAc group(s), N-acetylglucosamine is bound to the non-reducingterminal(s) of the Galβ1-4Glc or Galβ1-4GlcNAc group(s) throughβ1,3-linkage. Thus, the enzyme according to the present invention may beused for modification of sugar chains of glycoproteins and for synthesisof saccharides. Further, by administering this enzyme as an immunogen toan animal, an antibody to this enzyme may be prepared, so that theenzyme may be measured by an immunoassay using the antibody. Therefore,the enzyme according to the present invention and the nucleic acidcoding for the enzyme are useful for the preparation of such animmunogen. Such an antibody and the above-described nucleic acid formeasurement are useful for the measurement of the enzyme in the body,and the measurement is useful for the diagnoses, therapies andpreventions of cancers, immune diseases and infectious diseases bypylori.

The antibody, preferably the monoclonal antibody, which reacts with theenzyme of the present invention by antigen-antibody reaction, may beprepared by a well-known method comprising administering the enzyme ofthe present invention as an immunogen to an animal. Such an antibody maybe used for the diagnoses of cancers or tumors, preferably cancers ortumors of digestive organs, especially cancer or tumor of colon, morepreferably, for the diagnosis of colon cancer. In cases where theantibody is used for the diagnosis of a cancer or tumor, theabove-described enzyme is measured by an immunoassay utilizing theantigen-antibody reaction between the enzyme in the sample cells and theantibody, and the result is compared with the measurement resultsobtained for normal cells. If the measured amount of the enzyme issmaller than that in the normal cells, especially if the enzyme is notdetected, it is judged that the possibility that the sample is a canceror tumor is high. The immunoassays per se are well-known, and any of thewell-known immunoassays may be employed. That is, classifying the knownimmunoassays according to the reaction type, known immunoassays includesandwich immunoassays, competition immunoassays, agglutinationimmunoassays, Western blot and the like. Classifying the knownimmunoassays according to the label employed, known immunoassays includefluorescence immunoassays, enzyme immunoassays, radio immunoassays,biotin immunoassays and the like. Any of these immunoassays may beemployed. Further, diagnosis may be attained by immunohistostaining. Incases where a labeled antibody is used in the immunoassay, the methodsper se for labeling an antibody are well-known, and any of thewell-known methods may be employed. It is known that by decomposing anantibody with papain or pepsin, an antibody fragment such as Fabfragment or F(ab′)₂ fragment having the binding ability with thecorresponding antigen (such a fragment is called “antigen-bindingfragment” in the present specification) is obtained. The antigen-bindingfragments of the antibody of the present invention may also be used inthe same manner as the antibody.

These immunoassays per se are well-known in the art, and so it is notnecessary to explain these immunoassays in the present specification.Briefly, in sandwich immunoassays, for example, the antibody of thepresent invention or an antigen-binding fragment thereof is immobilizedon a solid phase as a first antibody. The first antibody is then reactedwith a sample, and after washing the solid phase, the resultant is thenreacted with a second antibody which reacts with the enzyme of thepresent invention by antigen-antibody reaction. After washing the solidphase, the second antibody bound to the solid phase is measured. Bylabeling the second antibody with an enzyme, fluorescent substance,radioactive substance, biotin or the like, measurement of the secondantibody bound to the solid phase may be attained by measuring thelabel. The above-mentioned measurement is conducted for a plurality ofstandard samples each containing a known concentration of the enzyme,and the relationship between the concentrations of the enzyme in thestandard samples and the measured amounts of the label is plotted toprepare a calibration curve. The enzyme in a test sample may bequantified by applying the measured amount to the calibration curve. Itshould be noted that the above-mentioned first antibody and theabove-mentioned second antibody may be exchanged. In agglutinationimmunoassays, the antibody according to the present invention or anantigen-binding fragment thereof is immobilized on particles such aslatex particles, and the particles are reacted with a sample, followedby measurement of the absorbance. The above-mentioned measurement isconducted for a plurality of standard samples each containing a knownconcentration of the enzyme, and the relationship between theconcentrations of the enzyme in the standard samples and the measuredabsorbance is plotted to prepare a calibration curve. The enzyme in atest sample may be determined by applying the measured absorbance to thecalibration curve.

The reagents necessary for each type of immunoassay are also well-knownin the art. Except for the antibody used, the immunoassay according tothe present invention may be carried out using an ordinary kit forimmunoassay. For example, such an immunoassay kit may usually includebuffer solution, solid phase, labeled second antibody and the like.

As will be concretely described in the Examples below, it was confirmedthat diagnoses of cancers and/or tumors can be attained by using theamount of expression of the enzyme of the present invention as an index.Thus, the present invention also provides a method for diagnosis of acancer or tumor, comprising determining the amount of expression of thegene coding for the enzyme of the present invention, in (a) samplecell(s) separated from body. As will be concretely described in theExamples below, the tumors which can be detected by the diagnosis methodaccording to the present invention are cancers or tumors for whichcancers are strongly suspected. As the sample cells, cells of digestiveorgans are preferred, and cells from colon are especially preferred. Byapplying the diagnosis method to these cells, cancers or tumors ofdigestive organs, especially cancer and/or tumor of colon may bediagnosed. The expression amount of the gene may be measured bymeasuring the amount of the mRNA transcribed from the gene or the amountof the cDNA prepared by using the mRNA as a template, or by measuringthe enzyme produced in the sample cells by an immunoassay using theantibody of the present invention. The measurement of the mRNA or cDNAmay be carried out using the above-described nucleic acid formeasurement according to the present invention by the method describedabove.

EXAMPLES

The present invention will now be described by way of Examples. However,the present invention is not restricted to the Examples. In thefollowing description, the nucleic acid having the nucleotide sequenceshown in SEQ ID NO:5, for example, may also be referred to as “SEQ IDNO:5” for convenience.

1. Search of Gene Database and Determination of Nucleotide Sequence ofβ3GnT-7

Using analogous genes which are knownβ1,3-N-acetylglucosaminyltransferase genes, β1,3-galactosyltransferasegenes and β1,3-N-acetylgalactosaminyltransferase gene, search ofanalogous genes was carried out on a gene database. The used sequenceswere β1,3-N-acetylglucosaminyltransferase genes with accession Nos.:AB049584, AB049585, AB049586 and AB045278; β1,3-galactosyltransferasegenes of accession Nos. AF117222, Y15060, Y15014, AB026730, AF145784 andAF145784; and β1,3-N-acetylgalactosaminyltransferase gene with accessionNo. Y15062 (all of the accession Nos. are of GenBank). The search wascarried out using a program tBlastn of BLAST, and all of the amino acidsequences corresponding to ORFs (Open Reading Frames) were included inthe search.

As a result, EST sequences with GenBank Accession Nos. AK000770 and ahuman genomic sequence AC017104 were discovered. Thus, using AC017104, alibrary was screened.

The used sample was human antrum cDNA library prepared by a conventionalmethod (Yuzuru Ikehara, Hisashi Narimatsu et al, Glycobiology vol. 9 no.11 pp. 1213–1224, 1999). The screening was carried out by a usualnucleic acid probe method using a radio isotope. The concrete procedureswere as follows:

First, using the λ phage prepared from a human antrum cDNA library by aconventional method as templates, PCR was performed using as primersCB-635(5′-cagca gctgc tggcc tacga agac-3′) (nt6814–6837 in AC017104) andCB-638 (5′-gcaca tgccc agaaa gacgt cgtc-3′) (nt7221–7245). The amplifiedDNA fragment having a size of about 430 bp was labeled with ³²P-dCTPusing Multiple DNA labeling system produced by AMERSHAM.

Using this probe, single plaques which hybridized with this probe werepicked up from the plaques of λ phage formed on E. coli. Existence ofthe target DNA region was confirmed by PCR using the above-mentionedprimers CB635 and CB638. Since the phage obtained from the plaques, inwhich the insertion of the DNA fragment was confirmed was constructed byλ ZAP II vector (STRATAGENE) (Yuzuru Ikehara, Hisashi Narimatsu et al,Glycobiology vol. 9 no. 11 pp. 1213–1224, 1999), a cDNA clone insertedinto pBluescript SK vector can be prepared (excision) by the methodaccording to the manufacturer's instruction. The recombinant vector wasprepared by this method, and a DNA was obtained from the obtainedcolony. The cDNA clone was then sequenced (SEQ ID NO:6).

The SEQ ID NO:6 obtained by the above-described method corresponded tont4828–7052 of AC017104 and lacked the 3′ region of ORF. Therefore, the3′ region was cloned after amplification thereof by PCR using the cDNA,and was ligated. That is, a primer CB-625 (5′-cgttc ctggg cctca gtttcctag-3′) (nt7638–7661) corresponding to a region downstream of thetermination codon was designed based on the sequence expected fromAC017104 resulted from the search by computer, and using this primer incombination with the above-described CB635, a DNA fragment was obtainedfrom the above-described human antrum cDNA library. The obtained DNAfragment was sequenced by a conventional method to obtain SEQ ID NO:7(nt6814–7661 in AC017104) (hereinafter referred to as “SEQ ID NO:3”). Bycombining this with SEQ ID NO:6, a theoretical ORF of 978 bp(nt6466–7452 in AC017104) was obtained, and a sequence of 328 aminoacids was deduced from this ORF, which was named β3GnT-7 (SEQ ID NO:8).It is known that glycosyltransferases are, in general, type 2 enzymeshaving one transmembrane segment. However, no hydrophobic region wasfound in the N-terminal region of this ORF sequence. Since it has beenreported that β1,3-N-acetylglucosaminyltransferase activity is detectedin human serum (Human Serum Contains N-Acetyllactosamine:β1,3-N-Acetylglucosaminyltransferase Activity. Hosomi, O., Takeya, A.,and Kogure, T. J. Biochem. 95, 1655–1659(1984)), the enzyme encoded bythis ORF was a secretory type enzyme having no transmembrane region.

To show that the ORF having the sequence shown in SEQ ID NO:8 and theamino acid sequence encoded thereby actually exist and function (i.e.,expressed), existence of the mRNA was checked by RT-PCR and confirmationof the PCR product by a restriction enzyme, and by direct sequencing(usual method) of the PCR product was carried out. As a result, it wasconfirmed that the above-described theoretical ORF surely existed andactually functioned.

As mentioned above, although it is known that glycosyltransferases are,in general, type 2 enzymes having one transmembrane segment, there is nohydrophobic region in the N-terminal region of the amino acid sequenceshown in SEQ ID NO:8, so that the enzyme was thought to be differentfrom the usual glycosyltransferases. Thus, whether a splicing varianthaving a hydrophobic region (transmembrane segment) in the N-terminalregion exists or not was checked by analyzing the nucleotide sequence inthe 5′ region (i.e., the N-terminal region of the amino acid sequence).

First, using Human stomach Marathon-Ready cDNA (CLONETECH), 5′-RACE(Rapid amplification of cDNA ends) was performed. More particularly,using the AP1 primer included in Marathon cDNA (an adaptor AP1 wasattached to the both ends of the DNA fragment, and an adaptor AP2 wasattached to the both inner ends thereof) and a primer β3GnT-7RACE-5(5′-GACCG ACTTG ACAAC CACCA GCA-3′) corresponding to the found sequenceregion, PCR was performed (94° C. for 60 seconds, 5 cycles of 94° C. for30 seconds–72° C. for 3 minutes, 5 cycles of 94° C. for 30 seconds–70°C. for 3 minutes, and 25 cycles of 94° C.–68° C. for 3 minutes) wasperformed. The obtained DNA product was subjected to nested PCR (94° C.for 60 seconds, 5 cycles of 94° C. for 30 seconds–72° C. for 3 minutes,5 cycles of 94° C. for 30 seconds–70° C. for 3 minutes, and 15 cycles of94° C.–68° C. for 3 minutes) using the AP2 primer included in MarathoncDNA and a primer β3GnT-7RACE-4 (5′-GTAGA CATCG CCCCT GCACT TCT-3′). Theobtained product was cloned into pGEMeasy (CLONETECH) and sequenced. Asa result, the sequence upstream of the initiation codon of the earlierdiscovered SEQ ID NO:6 was obtained, and a transmembrane region wasobserved when deduced into amino acid sequence. However, although the 5′region of the nucleotide sequence in the vicinity of the transmembraneregion was analyzed, the initiation codon of the ORF was not found.

Thus, using GeneScan, HMMgene and the like which were softwares foranalyzing gene regions, the translation region of the human genomicsequence AC017104 containing β3GnT-7 was analyzed. As a result, a firstexon of 11 bases (about 3 amino acid) (nt4331–4341 of AC017104)containing the initiation codon was expected. Thus, using a primercorresponding to an upstream region of the initiation codon, PCR wasperformed in order to determine whether the expected region existed as atranscript.

More particularly, PCR (30 cycles of 95° C. for 30 seconds, 60° C. for30 seconds, 72° C. for 60 seconds) was performed using as primersβ3GnT-7RACE-8 (5′-GCCCA GAGCT GCGAG CCGCT-3′) (nt4278–4300 in AC017104)and CB-638 (5′-GCACA TGCCC AGAAA GACGT CG-3′)((nt7224–7245 in AC017104),as a template Human leukocyte Marathon-Ready cDNA, and LA-Taq (TaKaRa).As a result, an amplification product having a size of 1046 bases wasobtained. This PCR product was purified and sequenced. It was proved, asexpected from the above-described analysis of the translation region,the 3′-side (nt4341) in the first exon was ligated to nt6258 in adownstream region.

By combining SEQ ID NOs: 6 and 7 and this result, the nucleotidesequence having 1206 bases shown in SEQ ID NO:5 and the amino acidsequence having 401 amino acids shown in SEQ ID NO:9 were obtained. TheSEQ ID NO:5 was one in which the upstream regions of 219 bases (73 aminoacids) (nt4331–4341 and nt6258–6465 in AC017104) were ligated to SEQ IDNO:8 (combination of SEQ ID NOs:6 and 7), and it was thought thatnt4342–6257 was spliced. Since SEQ ID NO:5 contains a transmembranesegment (nt6265–6322 in AC017104), SEQ ID NO:5 and SEQ ID NO:8 werethought to be the transmembrane type and secretory type having the sameactivity, respectively.

2. Insertion of β3GnT-7 into Expression Vector

To examine the activity of β3GnT-7, β3GnT-7 was expressed in insectcells. Although it is thought that the activity may be confirmed enoughby expressing the active region from the 119th amino acid to theC-terminal of SEQ ID NO:9, which region is relatively well conserved inthe other genes of the same family, the active region from the 75thamino acid to the C-terminal of β3GnT-7 (SEQ ID NO:9) was expressed.

The gene was incorporated into pFastBac of Gateway system fromINVITROGEN, and then a Bacmid by Bac-to-Bac system from INVITROGEN wasprepared.

{circle around (1)} Preparation of Entry Clone

PCR was performed using β3GnT-7S primer (5′-GGGGA CAAGT TTGTA CAAAAAAGCA GGCTT Cgcct ctcag gggcc ccagg cct-3′) and β3GnT-7A primer(5′-GGGGA CCACT TTGTA CAAGA AAGCT GGGTC catgg gggct cagga gcaag tgcc-3′)(the nucleotides shown in capital letters were the added sequence attLfor GATEWAY hereinbelow described), and as a template the DNA of β3GnT-7clone (the clone containing the theoretical ORF sequence) generated fromthe cDNA clone obtained by the screening and the DNA fragment obtainedby PCR, to obtain an amplification product.

This product was incorporated into pDONR201 by BP clonase reaction toprepare an “entry clone”. The reaction was carried by incubating amixture of 5 μl of the desired DNA fragment, 1 μl (150 ng) of pDONR201,2 μl of reaction buffer and 2 μl of BP clonase mix at 25° C. for 1 hour.After adding 1 μl of Proteinase K, the reaction mixture was left tostand at 37° C. for 10 minutes, thereby terminating the reaction.

Then the whole mixture (11 μl) was mixed with 100 μl of competent cells(E. coli DH5α), and after heat shock, the mixture was plated on an LBplate containing kanamycin. On the next day, colonies were collected,and existence of the desired DNA was directly confirmed by PCR. Fordouble check, the nucleotide sequence of the DNA was confirmed, andvector (pDONR-β3Gn-T7) was extracted and purified.

{circle around (2)} Preparation of Expression Clone

The above-described entry clone has attL at the both ends of theinserted region, the attL being a recombination site used when λ phageis cut out from E. coli. By mixing the entry clone with LR clonase (amixture of recombination enzymes Int, IHF and Xis of λ phage) and adestination vector, the inserted region is transferred to thedestination vector so that an expression clone is prepared. Theseoperations will now be described in detail.

Firstly, a mixture of 1 μl of the entry clone, 0.5 μl (75 ng) of pFBIF,2 μl of LR reaction buffer, 4.5 μl of TE and 2 μl of LR clonase mix wereallowed to react at 25° C. for 1 hour, and then 1 μl of Proteinase K wasadded, followed by incubation at 37° C. for 10 minutes, therebyterminating the reaction (by this recombination reaction, pFBIF-β3Gn-T7is generated). The pFBIF was one obtained by inserting Igκ signalsequence (MHFQVQIFSFLLISASVIMSRG) and FLAG peptide (DYKDDDDK) forpurification. The Igκ signal sequence was inserted in order to changethe expressed protein to a secretory protein, and the FLAG peptide wasinserted for purification. The DNA fragment obtained by PCR using as atemplate OT3 (5′-gatca tgcat tttca agtgc agatt ttcag cttcc tgcta atcagtgcct cagtc ataat gtcac gtgga gatta caagg acgac gatga caag-3′), andusing primers OT20 (5′-cgggatccat gcattttcaa gtgcag-3′) and OT21(5′-ggaat tcttgt catcg tcgtc cttg-3′) was inserted using Bam HI and EcoRI. Further, to insert the Gateway sequence, Conversion cassette wasinserted using Gateway Vector Conversion System (INVITROGEN).

Then the whole mixture (11 μl) was mixed with 100 μl of competent cells(E. coli DH5α), and after heat shock, the mixture was plated on an LBplate containing ampicillin. On the next day, colonies were collected,and existence of the desired DNA was directly confirmed by PCR, followedby extraction and purification of the vector (pFBIF-p3Gn-T7).

{circle around (3)} Preparation of Bacmid by Bac-to-Bac System

Using Bac-to-Bac system (INVITROGEN), recombination was carried outbetween the above-described pFBIF- and pFastBac, and G10 and othersequences were inserted into a Bacmid which was able to replicate ininsect cells. With this system, the desired gene is incorporated intothe Bacmid by the recombinant protein produced by a helper plasmid, onlyby incorporating pFastBac into which the desired gene was inserted,using the recombination site of Tn7 into an E. coli (DH10BAC) containingthe Bacmid. The Bacmid contains lacZ gene, so that classical selectionbased on the color, that is, blue (no insertion) or white (withinsertion), of the colony can be attained.

That is, the above-described purified vector (pFBIH-β3GnT-7) was mixedwith 50 μl of competent cells (E. coli DH10BAC), and after heat shock,the mixture was plated on an LB plate containing kanamycin, gentamycin,tetracycline, Bluo-gal and IPTG. On the next day, white single colonywas further cultured and Bacmid was collected.

3. Introduction of Bacmid into Insect Cells

After confirming that the desired sequence was inserted into the Bacmidobtained from the white colony, the Bacmid was introduced into insectcells Sf21 (commercially available from INVITROGEN). That is, to a 35 mmPetri dish, Sf21 cells in an amount of 9×10⁵ cells/2 ml (Sf-900SFM(INVITROGEN) containing an antibiotic) were added, and the cells werecultured at 27° C. for 1 hour to adhere the cells. (Solution A): To 5 μlof the purified Bacmid DNA, 100 μl of Sf-900SFM (INVITROGEN) notcontaining an antibiotic was added. (Solution B): To 6 μl of CelIFECTINReagent (INVITROGEN), 100 μl of Sf-900SFM (INVITROGEN) not containing anantibiotic was added. Solution A and Solution B were then gently mixedand the mixture was incubated for 15 to 45 minutes at room temperature.After confirming that the cells adhered, the culture medium wasaspirated and 2 ml of Sf-900SFM (INVITROGEN) not containing anantibiotic was added. To a solution (lipid-DNA complexes) prepared bymixing Solution A and Solution B, 800 μl of Sf900II not containing anantibiotic was added and the resultant was gently mixed. The culturemedium was aspirated, and diluted lipid-DNA complexes solution was addedto the cells, followed by incubating the cells at 27° C. for 5 hours.Thereafter, transfection mixture was removed and 2 ml of culture mediumSf-900SFM (INVITROGEN) containing an antibiotic was added, followed byincubating the resultant at 27° C. for 72 hours. Seventy two hours afterthe transfection, the cells were peeled off by pipetting, and the cellsand the culture medium were collected. The cells and the culture mediumwere centrifuged at 3000 rpm for 10 minutes, and the obtainedsupernatant was stored in a separate tube (this supernatant is theprimary virus solution).

To a T75 culture flask, Sf21 cells in an amount of 1×10⁷ cells/20 ml ofSf-900SFM (INVITROGEN) (containing an antibiotic) were placed, and theresultant was incubated at 27° C. for 1 hour. After the cells adhered,800 μl of the primary virus was added and the resultant was cultured at27° C. for 48 hours. Forty eight hours later, the cells were peeled offby pipetting and the cells and the culture medium were collected. Thecells and the culture medium were centrifuged at 3000 rpm for 10minutes, and the obtained supernatant was stored in a separate tube(this supernatant was used as the secondary virus solution).

Further, to a T75 culture flask, Sf21 cells in an amount of 1×10⁷cells/20 ml of Sf-900SFM (INVITROGEN) (containing an antibiotic) wereplaced, and the resultant was incubated at 27° C. for 1 hour. After thecells adhered, 1000 μl of the secondary virus solution was added and theresultant was cultured at 27° C. for 72 to 96 hours. After theculturing, the cells were peeled off by pipetting and the cells and theculture medium were collected. The cells and the culture medium werecentrifuged at 3000 rpm for 10 minutes, and the obtained supernatant wasstored in a separate tube (this supernatant was used as the tertiaryvirus solution). Further, to a 100 ml spinner flask, 100 ml of Sf21cells at a population of 6×10⁵ cells/ml was placed, and 1 ml of thetertiary virus solution was added, followed by culturing the cells at27° C. for about 96 hours. After the culturing, the cells and theculture medium were collected. The cells and the culture medium werecentrifuged at 3000 rpm for 10 minutes, and the obtained supernatant wasstored in a separate tube (this supernatant was used as the quaternaryvirus solution).

The primary to tertiary cell pellets were sonicated (sonication buffer:20 mM HEPES pH7.5, 2% Triton X-100 (trademark)) and the crude cellextract was 20-fold diluted with H₂O. The resultant was subjected toSDS-PAGE and then to Western blotting using anti-FLAG M2-peroxidase(A-8592, SIGMA) in order to confirm the expression of β3Gn-T7 protein.As a result, a plurality of broad bands (thought to be due todifferences in post-translational modifications by sugar chains or thelike) centering at the position of about 38–40 kDa were detected, sothat the expression was confirmed.

4. Resin Purification of β3Gn-T7

To 10 ml of the supernatant of FLAG-β3Gn-T7 of the quaternary infection,NaN₃ (0.05%), NaCl (150 mM), CaCl₂ (2 mM), and anti-M1 resin (SIGMA) (50μl) were added and the resulting mixture was stirred overnight at 4° C.On the next day, the mixture was centrifuged (3000 rpm for 5 minutes, at4° C.) and the pellet was collected. To the pellet, 900 μl of 2 mMCaCl2•TBS was added and the resultant was centrifuged again (2000 rpmfor 5 minutes, at 4° C.), and the pellet was suspended in 200 μl of 1 mMCaCl2•TBS to obtain a sample (β3GnT-7 enzyme solution) for themeasurement of activity.

5. Search of Acceptor Substrate of β3Gn-T7

As a result of molecular evolutionary analysis comparing β3Gn-T7 withβ1,3-N-acetylglucosaminyltransferases and β1,3-galactosyltransferases,β3Gn-T7 was classified into β1,3-N-acetylglucosaminyltransferases. Thus,firstly, analysis was performed using UDP-GlcNAc as the donor substrate.

Using the following reaction systems, the acceptor substrate wassearched. As the “acceptor substrate” in the reaction solution describedbelow, each of the following was used and whether each of themfunctioned as the acceptor or not was investigated: pNp-α-Glc,pNp-β-Glc, pNp-α-GlcNAc, pNp-β-GlcNAc, pNp-α-Gal, pNp-β-Gal,pNp-α-GalNAc, Bz-α-GalNAc, pNp-α-Xyl, pNp-β-Xyl, pNp-α-Fuc, Bz-α-Man,Bz-α-ManNAc, LacCer, GalCer typel and Bz-β-lactoside (all of them arefrom SIGMA) and Galβ1-4GlcNAc-α-pNp (TRONTO RESEARCH CHEMICAL).

The reaction solution (the numbers in the parentheses indicate the finalconcentrations) contained acceptor substrate (10 nmol), sodiumcacodylate buffer (pH7.2) (50 mM), Triton CF-54 (trademark) (0.4%),MnCl₂ (10 mM), UDP-GlcNAC (480 μM) and UDP-[¹⁴C]GlcNAC (175 nCi) andCDP-colline (5 mM), to which 10 μl of the β3Gn-T7 enzyme solution andH₂O were added to attain a final volume of 25 μl.

The reaction mixture was allowed to react at 37° C. for 5 hours, andafter completion of the reaction, 200 μl of 0.1 M KCl was added,followed by light centrifugation and collection of the supernatant. Thesupernatant was passed through Sep-Pak plus C18 Cartridge (WATERS)equilibrated by washing once with 10 ml of methanol and then twice with10 ml of H₂O, so as to adsorb the substrate and the product in thesupernatant on the cartridge. After washing the cartridge twice with 10ml of H₂O, the adsorbed substrate and the product were eluted with 5 mlof methanol. The eluted solution was evaporated to dryness by blowingnitrogen gas while heating the solution with a heat block at 40° C. Tothe resultant, 20 μl of methanol was added, and the resulting mixturewas plotted on a TLC plate (HPTLC plate Silica gel 60: MERCK), anddeveloped using a developing solvent having the composition ofchloroform:methanol:water (containing 0.2% CaCl₂)=65:35:8. Afterdeveloping the mixture up to 5 mm from the top end of the TLC plate, theplate was dried and the intensity of the radioactivity taken in theproduct was measured using Bio Image Analyzer FLA3000 (FUJI PHOTO FILM).

As a result, it was proved that β3GnT-7 is aβ1,3-N-acetylglucosaminyltrasferase having an activity to transferGlcNAc to Bz-β-lactoside and Galβ1-4Glc(NAc)-α-pNp, that is, an enzymewhich transfers GlcNAc to the galactose at the non-reducing terminal ofGalβ1-4Glc(NAc)-R.

6. Measurement of β3GlcNAcT Activity to N-glycan

As the enzyme source, the expressed and purified recombinant enzyme (towhich the FLAG sequence is fused) was used as in the case mentionedabove. As the acceptor substrates, commercially available PA-bound sugarchain substrates (produced by TAKARA BIO) shown in Table 1 were used.The reaction was carried out in a mixture containing 14 mM sodiumcacodylate buffer (pH7.4), 0.4% Triton CF-54, 10 mM MnCl₂, 50 mMUDP-GlcNAc (donor substrate), 20 pmol of the acceptor substrate and 100ng of the enzyme protein solution at 37° C. for 16 hours. The reactionwas terminated at 95° C. for 3 minutes, and 80 μl of water was added.The resulting mixture was passed through Ultra-free MC column (WATERS),and 45 μl aliquot of the passed solution was subjected to HPLC. Theconditions of the HPLC were as described below. The conversion enzymeactivity (%) was determined using a solution which did not containUDP-GIcNAc (donor substrate) as a control. The results are shown inTable 1 below.

(HPLC Conditions)

-   Buffer I.a: 100 mM acetic acid/triethylamine, pH 4.0-   Buffer I.b: 100 mM acetic acid/triethylamine, pH 4.0 (containing    0.5% 1-butanol)-   gradient: 5–55%: Buf. I.b (0–60 min.),-   flow rate: 1.0 ml/min.-   column: PalPak Type R (TaKaRa Cat. No. CA8000)-   column oven temp: 40° C.-   HPLC System: Shimadzu LC-10AD vp, CTO-10AC vp DGU-14A, cell temp    controller-   Detector: Fluorescence: RF-10AXL UV: SPD-10Avp

TABLE 1 Con- version Activity Acceptor Substrate (%)

18.3

26.0

20.3

20.6

17.3

18.1

 0.0 Galβ1-4GlcNAcβ1-3Galβ1-4Glc-PA —7. Measurement of Expression of Enzyme by Flow Cytometry

The β3GnT-7(G10) gene was incorporated into pDEST12.2 vector(INVITROGEN) to prepare pDEST12.2-G10 vector DNA. More particularly,this was carried out as follows: Using primers described belowcontaining the sequence of the Gateway system of INVITROGEN, a cDNA fromColo205 cells (colon cancer cells) was amplified by PCR, and theamplification product was first incorporated into the pDONR vector by BPreaction. After confirming the DNA sequence by sequencing the vector,the insert was transferred from the pDONR vector to pDEST12.2 vector byLR reaction. These operations were carried out using the vectors andreagents contained in the kit of INVITROGEN in accordance with theinstructions included in the commercial product.

G10/ORF-F1 Primer

-   ggggacaagtttgtacaaaaaagcaggcttctggcgcccagagctgcgagccgct    (In this, ggggacaagtttgtacaaaaaagcaggcttc is a sequence in the    vector)    G10/ORF-R1 Primer-   ggggaccactttgtacaagaaagctgggtccatgggggctcaggagcaagtgcc    (In this, the cDNA sequence of b3GnT7 gene is from    catgggggctcaggagcaagtgcc) By the above-described procedures, a    recombinant vector was obtained in which a DNA fragment containing    the cDNA shown in SEQ ID NO:5 to which the region other than the    cDNA sequence in the above-described primers was attached to the 5′-    and 3′-ends thereof was inserted. This recombinant vector was    introduced into HCT15 cell line and LSC cell line (both are colon    cancer cell lines) by a conventional method. As a control, the    pDEST12.2 vector DNA in which the gene was not incorporated was    introduced into the cell lines in the same manner (Mock cells).    After carrying out the selection by 0.8 mg/ml of G418 (INVITROGEN)    for one month, the cells were harvested. The harvested cells were    washed twice with 1% BSA/0.1% NaN3/PBS(−). The cell population was    adjusted to 1×10⁷ cells/ml, and 100 μl (1×10⁶ cells) aliquot thereof    was used for one sample. After centrifugation, the supernatant was    removed and the resultant was diluted to a concentration of 10    μg/ml. To the resultant, 100 μl each of the FITC-labeled lectins    described below were added, and the cells were suspended. After    allowing the reaction at 4° C. in the dark (refrigerator) for 30    minutes, 100 μl of 1% BSA/0.1% NaN₃/PBS was added to each well to    carry out washing. The resultant was centrifuged at 1000 rpm for 5    minutes, and the supernatant was removed. The washing was repeated    once more. The resulting cells were suspended in 1 ml of 0.5%    paraformaldehyde/PBS to fix the cells, and analyzed by flow    cytometry FACSCalibur (BECTON DICKINSON) after passing the cells    through a nylon mesh. The results are shown in FIGS. 1–3.

The used lectins were Lycopersicon esculentum (LEA) and Triticum vulgare(WGA), both of which recognize the repetition of N-acetyl lactosaminestructure, and N-acetyl glucosamine structure, and labeled with FITC(purchased from HONEN, SEIKAGAKU CORPORATION, EY LABORATORIES and soon).

FIG. 1 shows the results of the flow cytometry showing the bindingproperty between the HCT15 colon cancer cell line and the LEA lectin.FIG. 2 shows the results of the flow cytometry showing the bindingproperty between the LSC colon cancer cell line transformed with therecombinant vector containing the gene of the present invention or thevector not containing the gene of the present invention and LEA lectin.FIG. 3 shows the results of the flow cytometry showing the bindingproperty between the HCT15 colon cancer cell line and the WGA lectin. Ineach of the drawings, the bold line shows the results of the cellstransformed with the recombinant vector containing β3GnT-7 gene, and thethin line shows the results of the cells (Mock cells) transformed withthe vector not containing β3GnT-7 gene.

As shown in FIGS. 1–3, in all of the cases, the fluorescence intensitywas shifted, which indicates that the N-acetyl lactosamine-containingstructure was increased in the cells into which the DNA of pDEST12.2-G00containing β3 GnT-7(G10) gene was incorporated.

8. Analysis of Tissue-specific Expression of β3GnT-7

The expression of the gene in tissues and in cell lines was examined byReal Time PCR method (Gibson, U. E., Heid, C. A., and Williams, P. M.(1996) Genome Res 6, 995–1001). Human tissue cDNAs used as materialswere the Marathon cDNAs. From the various cell lines, total RNAs wereextracted by a conventional method and the cDNAs were synthesized. Forobtaining the calibration curve of β3GnT-7, a plasmid containing β3GnT-7gene inserted in pDONR™201 vector DNA was used. As a control for theendogenous expression, constantly expressed humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH)) was used. Forobtaining the calibration curve of GAPDH, a plasmid containing the GAPDHgene in pCR2.1 (INVITROGEN) was used. As the primer set and probe forβ3GnT-7, the following were used: RT-β3GnT-7-F2;5′-TTCCTCAAGTGGCTGGACATC-3′, RT-β3GnT-7-R2;5′-GCCGGTCAGCCAGAAATTC-3′,probe; 5′-Fam ACTGCCCCCACGTCCCCTTCA-MGB-3′. As the primer set and probefor GAPDH, a kit (Pre-Developed TaqMan® Assay Reagents Endogenous HumanGAPDH (APPLIED BIOSYSTEMS) was used. The PCR was performed using TaqManUniversal PCR Master Mix (APPLIED BIOSYSTEMS) under the conditions of50° C. for 2 minutes, then at 95° C. for 10 minutes, and repeating 50cycles of 95° C. for 15 seconds–60° C. for 1 minute. The quantitation ofthe PCR product was carried out using ABI PRIAM7700 Sequence DetectionSystem (APPLIED BIOSYSTEMS). The expression amount of G11 was normalizedby dividing the amount by the amount of the transcription product of theconstantly expressed GAPDH. The results for the human tissues aresummarized in Table 2, and the results for the cell lines are summarizedin Table 3.

TABLE 2 Tissue β3GnT-7/GAPDH brain 0.01045 cerebral cortex 0.04522cerebellum 0.02345 fetal brain 0.02030 bone marrow 0.01462 thyroid0.04084 thymus 0.01274 spleen 0.10108 leukocyte 0.07876 heart 0.00956skeletal muscle 0.00071 lung 0.12146 liver 0.02299 esophagus 0.00605stomach 0.26922 small intestine 0.09333 colon 0.07630 pancreas 0.27317kidney 0.01161 adrenal 0.15069 mammary gland 0.02560 uterus 0.07747placenta 0.18763 ovary 0.11465 testis 0.05323

The tissues in which β3GnT-7 was highly expressed were pancreas,stomach, placenta and adrenal, and the tissues in which β3GnT-7 wasmoderately expressed were colon, leukocyte, lung, ovary, smallintestine, spleen, testis, uterus and cerebral cortex. In the tissuesother than these tissues, the expression amount was relatively low.

TABLE 3 Cell (origin) β3GnT-7/GAPDH GOTO (neuroblastoma) 0.00012 SCCH-26(neuroblastoma) 0.00137 T98G (glioblastoma) 0.00032 U251 (glioblastoma)0.00023 Leukemia (premyeloblastic leukemia) 0.35660 Melanoma (skin)0.01255 HL-60 (premyeloblastic leukemia) 0.17663 K562 (leukemia) 0.00038U937 (monocyte) 0.01617 Daudi (B cell (Burkitt's)) 0.00437 PC-1 (lung)0.00000 EBC-1 (lung) 0.00121 PC-7 (lung) 0.00017 HepG2 (liver) 0.01199A431 (esophagus) 0.01031 MKN45 (stomach) 0.00027 KATOIII (stomach)0.03964 HSC43 (stomach) 0.00031 Colo205 (colon) 0.00278 HCT15 (colon)0.00193 LSC (colon) 0.00003 LSB (colon) 0.00128 SW480 (colon) 0.00045SW1116 (colon) 0.13076 Capan-2 (pancreas) 0.03664 PA-1 (uterus) 0.00290

Expression of β3GnT-7 in cell lines was lower than that in normaltissues. In HL60 cells originated from premyeloblastic leukemia and inSW1116 cells originated from colon, the expression level was high.

It was easily thought that the expression amount of β3GnT-7 is changedwhen the degree of differentiation is changed by cancerization or thelike, so that there is a possibility that measurement of the expressionamount of β3GnT-7 may be used for diagnoses of diseases. Further, asdescribed above, there is a possibility that there are two initiationsites in β3GnT-7, so that there is a possibility that by measuring thechange of the splicing variants, the state of differentiation andpathological change of the cells may be measured.

9. Expression of β3GnT-7 Gene in Normal Tissues and Cancer Tissues ofColon Cancer Patients

The expression amounts of β3GnT-7 in normal (N)-tissues and cancer (T)tissues of actual colon cancer (DK) patients were measured by the methoddescribed in “8. Analysis of Tissue-specific Expression of β3GnT-7”. Theresults are shown in FIG. 4. From these results, in samples except forDK3, that is, in samples of DK10, DK15, DK19, DK22 and DK23, thetendency that expression of β3GnT-7 in cancer tissue is smaller than inthe normal tissue was observed.

1. An isolated protein having an amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having a homology of not less than 95% tothe amino acid sequence of SEQ ID NO:1, which has an activity totransfer N-acetylglucosamine to a non-reducing terminal of Galβ1-4Glc orGalβ1-4GlcNAc group through β1,3-linkage.
 2. The protein according toclaim 1, which has the amino acid sequence shown in SEQ ID NO: 3, orwherein the amino acid sequence has a homology of not less than 95% tosaid amino acid sequence shown in SEO ID NO:3.
 3. The protein accordingto claim 2, which has the amino acid sequence shown in SEQ ID NO:3.
 4. Aprotein comprising the amino acid sequence recited in claim 1 or claim2, which has an activity to transfer N-acetylglucosamine to anon-reducing terminal of Galβ1-4Glc or Galβ1-4GlcNAc group throughβ1,3-linkage.
 5. A method for diagnosis of a cancer and/or tumor,comprising determining the amount of said protein according to claim 3,in (a) sample cell(s) separated from body.
 6. The method according toclaim 5, wherein said sample cell(s) is(are) originated from a digestiveorgan, and wherein said method is for diagnosis of a cancer and/or tumorof the digestive organ.
 7. The method according to claim 6, wherein saidsample cell(s) is(are) originated from colon, and wherein said method isfor diagnosis of colon cancer.